Sequence alignment SEQ 1 VLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGKK VADALTNAVAHVDDPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHA SLDKFLASVSTVLTSKYR SEQ

Sequence alignment SEQ 1: VLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGKK VADALTNAVAHVDDPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHA SLDKFLASVSTVLTSKYR SEQ 2: VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVK AHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEF TPPVQAAYQKVVAGVANALAHKYH Are these two proteins alike?

Dotplots: visual sequence comparison 1. Place two sequences along axes of plot 1. Place dot at grid points where two sequences have identical residues 1. Diagonals correspond to conserved regions

Alignment scores: match vs. mismatch Simple scoring scheme (too simple in fact…): Matching amino acids: Mismatch: 0 5 Scoring example: K A W S A D V : : : K D W S A E V 5+0+5+5+5+0+5 = 25

Pairwise alignments: conservative substitutions 43. 2% identity; alpha beta Global alignment score: 374 10 20 30 40 50 V-LSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHF-DLS-----HGSA : : . : . : : : : . VHLTPEEKSAVTALWGKV--NVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNP 10 20 30 40 50 60 70 80 90 100 110 QVKGHGKKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHL. : : : : . . . . : : : : : . KVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHF 60 70 80 90 100 110 120 130 140 alpha PAEFTPAVHASLDKFLASVSTVLTSKYR : : : . : . . . : : . beta GKEFTPPVQAAYQKVVAGVANALAHKYH 120 130 140

Amino acid properties Serine (S) and Threonine (T) have similar physicochemical properties Aspartic acid (D) and Glutamic acid (E) have similar properties => Substitution of S/T or E/D occurs relatively often during evolution => Substitution of S/T or E/D should result in scores that are only moderately lower than identities

Protein substitution matrices A R N D C Q E G H I L K M F P S T W Y V 5 -2 -1 -1 -1 0 -2 -1 -1 -3 -1 1 0 -3 -2 0 A BLOSUM 50 matrix: 7 -1 -2 -4 1 0 -3 0 -4 -3 3 -2 -3 -3 -1 -1 -3 R 7 2 -2 0 0 0 1 -3 -4 0 -2 -4 -2 1 0 -4 -2 -3 N 8 -4 0 2 -1 -1 -4 -4 -1 -4 -5 -1 0 -1 -5 -3 -4 D 13 -3 -3 -2 -2 -4 -1 -1 -5 -3 -1 C • Positive scores on diagonal (identities) 7 2 -2 1 -3 -2 2 0 -4 -1 0 -1 -1 -1 -3 Q 6 -3 0 -4 -3 1 -2 -3 -1 -1 -1 -3 -2 -3 E 8 -2 -4 -4 -2 -3 -4 -2 0 -2 -3 -3 -4 G • Similar residues get higher (positive) scores 10 -4 -3 0 -1 -1 -2 -3 2 -4 H 5 2 -3 2 0 -3 -3 -1 4 I 5 -3 3 1 -4 -3 -1 -2 -1 1 L • Dissimilar residues get smaller (negative) scores 6 -2 -4 -1 0 -1 -3 -2 -3 K 7 0 -3 -2 -1 -1 0 1 M 8 -4 -3 -2 1 4 -1 F 10 -1 5 -1 2 5 -4 -4 -3 15 -3 -2 -2 2 8 -3 -2 0 -3 -1 P S T W Y 5 V

1 PLC. _ When Blast works! 1 PLB. _